Measurement of Available Carbohydrates in Cereal and Cereal Products, Dairy Products, Vegetables, Fruit, and Related Food Products and Animal Feeds: First Action 2020.07

Abstract Background The level of available carbohydrates in our diet is directly linked to two major diseases: obesity and Type II diabetes. Despite this, to date there is no method available to allow direct and accurate measurement of available carbohydrates in human and animal foods. Objective The aim of this research was to develop a method that would allow simple and accurate measurement of available carbohydrates, defined as non-resistant starch, maltodextrins, maltose, isomaltose, sucrose, lactose, glucose, fructose, and galactose. Method Non-resistant (digestible) starch is hydrolyzed to glucose and maltose by pancreatic α-amylase (PAA) and amyloglucosidase at pH 6.0 with shaking or stirring at 37°C for 4 h. Sucrose, lactose, maltose, and isomaltose are completely hydrolyzed by specific enzymes to their constituent monosaccharides, which are then measured using pure enzymes in a single reaction cuvette. Results A method has been developed that allows the accurate measurement of available carbohydrates in all cereal, vegetable, fruit, food, and feed products, including dairy products. Conclusions A single-laboratory validation was performed on a wide range of food and feed products. The inter-day repeatability (RSDr, %) was <3.58% (w/w) across a range of samples containing 44.1–88.9% available carbohydrates. The LOD and LOQ obtained were 0.054% (w/w) and 0.179% (w/w), respectively. The method is all inclusive, specific, robust, and simple to use. Highlights A unique method has been developed for the direct measurement of available carbohydrates, entailing separate measurement of glucose, fructose, and galactose, information of value in determining the glycemic index of foods.

that time, as detailed by Southgate (2). In the United States, the "by difference" method for determining total carbohydrates was introduced by Atwater and Woods in 1896 (3) and is still in use today. The moisture, protein, fat, and ash content of a food are determined and then subtracted from the total weight of the food and the remainder, or "difference," is considered to be total carbohydrate. More recently, "net carbohydrates" has been determined by subtracting dietary fiber from the total carbohydrates value. This value is meant to equate with "available carbohydrates" determined directly, but, the "by difference" figure includes non-carbohydrate components such as lignin, organic acids, tannins, waxes, and some Maillard products. Also, it combines all the analytical errors from the other analyses (4). To date, methods for the direct measurement of available carbohydrates have been difficult to implement and are non-specific, as detailed by Southgate (2).
In the measurement of available carbohydrates, total starch has traditionally been measured instead of just digestible starch, and sucrose hydrolysis was achieved with invertase (b-fructofuranosidase). Englyst et al. (5) developed methodology for the measurement of digestible starch and resistant starch and this methodology is widely used and cited. These authors considered that digestion of starch by pancreatic a-amylase (PAA) plus amyloglucosidase (AMG) was complete within 2 h of incubation at 37 C. However, since literature indicate that the average time of residence of food in the human small intestine is $ 4 h (6, 7), we have settled on this incubation time in developing the current methodology. Starch that is digested during this time period is part of the carbohydrate that is absorbed in the human small intestine. Traditionally, the measurement of sucrose has involved hydrolysis to glucose and fructose by invertase, with subsequent measurement of the glucose and fructose. However, invertase also hydrolyzes the lower degree of polymerization (DP) fructooligosaccharides (FOS), resulting in overestimation of sucrose in those samples that contain FOS. In the current study, maltose is hydrolyzed to glucose by maltase, and sucrose is specifically hydrolyzed to glucose and fructose by a sucrase enzyme that has no action on FOS (7,8). Lactose is hydrolyzed to glucose and galactose by a specific b-galactosidase (MZ104). This b-galactosidase also has some action on GOS, if present in the sample, leading to an overestimation of available carbohydrates for such samples. Isomalto-oligosaccharides (IMO) find some application in food preparations as a digestion-resistant carbohydrate. However, in the presence of the PAA/AMG used in this method, IMO are hydrolyzed mainly to glucose and isomaltose, an oligosaccharide that is digested in the human small intestine (7,9,10). Consequently, in the current procedure, isomaltose is hydrolyzed to glucose using oligo-1,6-a-glucosidase. Galactose, glucose, and fructose are measured employing high-purity and specific enzymes. This method is an extension of AOAC Method 2017.16 (7,8) for the measurement of total dietary fiber. Incubation conditions with PAA/AMG are the same, however scaled down two-fold. Aliquots of the hydrolysate are incubated with sucrase, maltase, and b-galactosidase and total released galactose, glucose, and fructose are measured. The method is commercially available as the Available Carbohydrates Assay Kit (Megazyme Cat. No. K-AVCHO).
A. Method (a) Scope of the method.-(1) Target analyte.-Digestible starch, maltodextrins, maltose, IMO, isomaltose, sucrose, lactose, glucose, fructose, and galactose. The specific b-galactosidase (MZ104) employed in this method to hydrolyze lactose also has some action on specific GOS and thus could lead to an overestimation of available carbohydrates in samples containing GOS.  Test solutions are then removed, diluted, and centrifuged. Aliquots of this diluted solution are incubated with a mixture of sucrase, maltase, and b-galactosidase at pH 6.5 and 30 C for 30 min, during which time sucrose is specifically hydrolyzed to glucose and fructose by sucrase enzyme, maltose is hydrolyzed to glucose, and lactose is hydrolyzed to galactose and glucose by b-galactosidase (Equations 2-4).
( In the presence of the enzymes galactose dehydrogenase (GalDH) and galactose mutarotase (GalM), b-D-galactose is oxidized by nicotinamide-adenine dinucleotide phosphate (NADP þ ) to D-galactonate with the formation of reduced nicotinamideadenine dinucleotide phosphate (NADPH) (Equation 6). (6) (GalDH/GalM, pH 7.6, 30°C, 3 min) b-D-Galactose + NADP + galactonate + NADPH + H + The amount of NADPH formed in this reaction is stoichiometric with the amount of D-galactose. It is the NADPH which is measured by the increase in absorbance at 340 nm.
The method is simple to use and the absorbance response for galactose, glucose, and fructose is the same ( Figure  2020.07A).     Preparations of reagents and buffers which meet the criteria as specified in the method above may also be used.

E. Safety Considerations
The general safety measures that apply to all chemical substances should be adhered to. For more information regarding the safe use and handling of the Available Carbohydrates Assay Kit reagents (K-AVCHO) refer to the K-AVCHO SDS document that is downloadable from where the product appears on the Megazyme website (www.megazyme.com). Some individuals are allergic to powdered PAA and/or amyloglucosidase. This enzyme preparation should be weighed and dissolved in a wellventilated fume cupboard. The preparation can be stabilized with ammonium sulphate to reduce handling of the powder product.

F. Preparation of Test Materials
Collect and prepare food materials as "intended to be eaten," i.e., cook pasta and potatoes. For dry foods, animal feeds, and breakfast cereals, grind approximately 50 g material in a grinding mill to pass a 0.5 mm sieve. Transfer all materials into widemouthed plastic jars, and seal and mix well by shaking and inversion. Freeze dry high-moisture-(>25% w/w) containing materials. Pour canned beans and vegetables onto a strainer and wash with demineralized water, freeze dry, and mill to pass a 0.5 mm screen. Homogenize dried fruit materials in a highspeed blender (e.g., Nutri-Bullet) and then dry the paste in a forced air oven at 40 C overnight. Further grind materials to be analyzed using a mortar and pestle. Homogenize high-fatcontaining materials such as malted milk biscuits, chocolate digestive biscuits, jam and cream biscuits, shortbread finger biscuits, and Cadbury V R dairy milk chocolate using a high-speed blender (e.g., Nutri-Bullet). In a well-ventilated fume cupboard, transfer a portion of the homogenized material (approximately 10 g, weighed accurately) to a pre-weighed 200 mL beaker and add 50 mL of petroleum ether. Stir the mixture with a spatula for 20 s and allow the solids to settle. Carefully decant the supernatant and then repeat this process a further two times. Allow the solids in the beaker to dry in a well-ventilated fume hood and weigh. Calculate fat content. Store materials in the presence of a desiccant.

G. Measurement of Enzyme Activities
Measure the activity of a-amylase activity in PAA using the Ceralpha V R assay procedure employing non-reducing endblocked p-nitrophenyl maltoheptaoside in the presence of excess levels of thermostable a-glucosidase. Perform incubations in sodium maleate buffer at pH 6.9 and 40 C as described in the a-Amylase Assay Kit (Ceralpha (11). One unit of enzyme activity is defined as the amount of enzyme that releases one mmole of p-nitrophenol per minute under the defined assay procedure. The a-amylase activity reported is that measured at the optimal pH of 6.9. However, incubations for the measurement of digestible starch, resistant starch, and available carbohydrates were performed at pH 6.0. a-Amylase activity at pH 6.0 is $77% of that at pH 6.9 (12). AMG was assayed by incubating 0.2 mL of suitably diluted enzyme in 100 mM sodium acetate buffer (pH 4.5) with 0.5 mL of soluble starch (10 mg/mL) in 100 mM sodium acetate buffer (pH 4.5) at 40 C. At various time intervals, reaction tubes were heated to $100 C in a boiling water bath to terminate the reaction and released glucose was measured using GOPOD reagent [Glucose Assay Kit (GOPOD Format); Megazyme Cat. No. K-GLUC]. One unit of AMG is defined as the amount of enzyme required to release one mmole of D-glucose per minute at pH 4.5 and 40 C. When in admixture with PAA, AMG was assayed using AMG Assay Reagent (Megazyme Cat. No. R-AMGR3) and units of activity on starch were calculated using a conversion factor. The AMG activity reported is that measured at the optimal pH of 4.5. However, incubations for the measurement of digestible starch, resistant starch, and available carbohydrates were performed at pH 6.0. AMG activity at pH 6.0 is $60% of that at pH 4.5.

I. Calculations
Determine the absorbance difference (A 2 -A 1 ) for both blank and sample. Subtract the absorbance difference of the blank from the absorbance difference of the sample, thereby obtaining DA galactose . Determine the absorbance difference (A 3 -A 2 ) for both blank and sample. Subtract the absorbance difference of the blank from the absorbance difference of the sample, thereby obtaining DA glucose .
Determine the absorbance difference (A 4 -A 3 ) for both blank and sample. Subtract the absorbance difference of the blank from the absorbance difference of sample, thereby obtaining DA fructose .
The values of DA galactose , DA glucose , and DA fructose should as a rule be at least 0.100 absorbance units to achieve sufficiently accurate results. If values are less than this, the possibility of decreasing the dilution of the sample extract or increasing the volume of the aliquot analyzed should be considered. This may not be possible if the combined absorbance then falls outside the analytical range of the assay. If the combined absorbance value is above 1.40, the sample should be diluted further and re-assayed.
The concentration of galactose, glucose and fructose can be calculated as follows:   If oligo-1,6-a-glucosidase (10 mL) is added to the incubation mixture, appropriate adjustments of the final incubation volumes need to be made (i.e., for galactose, the final volume will be 2.43 mL, for glucose it will be 2.45 mL, and for fructose it will be 2.47 mL). When analyzing solid and semi-solid samples which are weighed out for sample preparation, the content (g/100 g) is calculated from the amount weighed as follows: Content of galactose:  Note: These calculations can be simplified by using the Megazyme Mega-Calc TM , downloadable from where the product appears on the Megazyme website (www.megazyme.com).
Measurement of galactose, glucose, and fructose using specific enzymes and following the increase in the reduction of NADP þ to NADPH is shown in Figure 2020.07E.

J. Indicative Controls
Indicative controls are used as a check on assay conditions. Available carbohydrates, galactose, glucose, and fructose values for the included control sample should be within 3% of that stated on the control label. Inulin, levan, and galactosylsucrose oligosaccharides should not be hydrolyzed and thus should contribute nothing to the available carbohydrates value. Sucrose in FOS (e.g., Orafti P95 V R ) will be hydrolyzed by the sucrase and, thus, correctly measured as available carbohydrates.

Planning
The purpose of this report is to verify and validate a method for measurement of available carbohydrates, defined as the sum of digestible starch, maltodextrins, maltose, sucrose, lactose, glucose, fructose, and galactose (and isomaltose if present). Digestible starch, maltodextrins, maltose, sucrose, lactose, and isomaltose are hydrolyzed to glucose plus fructose and/or galactose and the individual monosaccharides are then specifically measured. This method is an extension of AOAC Method 2017.16 (rapid integrated method for measurement of dietary fiber). Digestible starch and maltodextrins are hydrolyzed to glucose with PAA plus amyloglucosidase and sucrose is hydrolyzed to glucose plus fructose with a specific sucrase enzyme; lactose is hydrolyzed to galactose and glucose; the remaining traces of maltose are hydrolyzed by maltase to glucose and isomaltose is hydrolyzed to glucose.
Working range.-The assay follows the Megazyme Available Carbohydrates Assay Kit method (Megazyme Cat. No. K-AVCHO) and has a working range of 0.18-100% (w/w) available carbohydrates in the sample. For samples containing 0-10% (w/w) available carbohydrates, after incubation of 500 mg of sample with PAA/AMG in 20.5 mL of buffer, an aliquot (1.0 mL) is added to 5 mL of water (diluted 6-fold) and mixed, before centrifugation and analysis. For samples containing 10-100% (w/w) available carbohydrates, the same incubation conditions are performed, but 1 mL of the incubation mixture is added to 25 mL of water (diluted 26-fold) and mixed before centrifugation and analysis. The linearity of the reactions employed to measure galactose, glucose, and fructose are shown in Figure 2020.07A. The r 2 values were 0.9999 for each sugar. Also, the absorbance response with the three sugars is identical.
Selectivity.-The Available Carbohydrates Assay Kit is specific for digestible starch plus maltodextrins, maltose, isomaltose, sucrose, lactose, glucose, fructose, and galactose, as described. In the presence of highly purified PAA plus amyloglucosidase, digestible starch is hydrolyzed to glucose plus traces of maltose, and IMO are hydrolyzed mainly to glucose and isomaltose. Remaining traces of maltose are hydrolyzed to glucose with maltase; sucrose is quantitatively hydrolyzed to glucose and fructose by a specific sucrase enzyme which has no action on inulin, FOS, levan, or galactosyl-sucrose oligosaccharides (e.g., raffinose); lactose is quantitatively hydrolyzed to galactose and glucose and isomaltose is hydrolyzed to glucose.
Specificity.-Levan, kestose, and raffinose are not hydrolyzed in the available carbohydrates assay procedure and thus do not add to the value determined. Commercial Orafti GR contains approximately 0.1% w/w fructose and 0.4% w/w sucrose. Orafti P95 preparation (FOS) contains approximately 5% w/w of fructose, glucose, and sucrose. Thus, an available carbohydrates value of approximately 5% w/w is obtained for Orafti P95 (Table 1). There is no hydrolysis of the FOS in this material. Confirmation of the lack of hydrolysis of Orafti P95 V R by sucrase, but rapid hydrolysis by invertase is shown in Figure 1. In these experiments, Orafti P95 or sucrose (10 mg) was incubated with either invertase (200 U, 1 mL, pH 4.5) or sucrase (40 U on sucrose, 1 mL at pH 6.5) at 40 C for various time intervals. Reactions were terminated by heating tubes in boiling water for 3 min and samples deionized and analyzed by HPLC on TSKgel V R G2500PW XL columns, 30 cm Â 7.8 mm, connected in series. Clearly, Orafti GR [ Figure 1(a)] is rapidly hydrolyzed by invertase [ Figure 1   This experiment demonstrates both the effectiveness and specificity of sucrase, with completely hydrolysis of sucrose but no hydrolysis of FOS. Galacto-oligosaccharides (GOS) find use in the food industry as a prebiotic and as digestion resistant oligosaccharide mixtures. The b-galactosidase (MZ104) employed in the current method gives complete hydrolysis of lactose with limited hydrolysis of allolactose and GOS of DP 3 and greater (13) under the assay conditions used. Further knowledge on the likely degree of hydrolysis of commercial GOS preparations by b-galactosidase MZ104 as used in the method described here will provide information on likely overestimation of available carbohydrates in samples that are rich in these oligosaccharides.
Isomalto-oligosaccharides find application in food products as a source of "digestion-resistant" carbohydrate. The chromatographic patterns of a commercially available isomaltooligosaccharide mixture, "Advantafiber V R ," both before and after hydrolysis by PAA/AMG for 1 and 4 h, under the conditions of AOAC Method 2017.16, are shown in Figure 2. After 4 h incubation with PAA/AMG, 60% of the oligosaccharides are converted to glucose, and 28% to isomaltose. Approximately 12% of the remaining oligosaccharides have a degree of polymerisation of !3 (i.e., only 12% can be defined as dietary fiber). This result is consistent with the $10% dietary fiber values obtained for this material using the Matsutani modification (AOAC Method 2001.03) of AOAC Method 985.29 (McCleary, unpublished), and published data showing that isomaltose is hydrolysed by the mucosal a-glucosidases of the small intestine (7,9,10). The isomaltose (disaccharide) that is produced under the incubation conditions of AOAC Method 2017.16 is not defined as dietary fiber. Also, it is not measured as available carbohydrates because it is not hydrolyzed to glucose under the incubation conditions described in Figure 2020.07D. Complete hydrolysis requires the use of another enzyme, namely, oligo-1,6-a-glucosidase to cleave the 1,6-a-glucosyl linkages in isomalto-oligosaccharides. Hydrolysis of sucrose, lactose, and also isomaltose can be achieved under the conditions defined in the available carbohydrates protocol (Figure 2020.07D), if 10 mL of oligo-1,6-a-glucosidase (1000 U/mL) (Megazyme Cat. No. E-OAGUM) is included (Figure 3).
Oligo-1,6-a-glucosidase is now included in the enzyme mixture used to hydrolyze sucrose, maltose, and lactose provided in the Megazyme Available Carbohydrates test kit (K-AVCHO) The mixture contains sucrase (170 U/mL), maltase (1000 U/mL), MGZ104 b-galactosidase (500 U/mL), and oligo-1,6-a-glucosidase (1000 U/mL) as an ammonium sulphate suspension. In this case, sample extract in water (0.1 mL) and sodium maleate buffer (pH 6.5) containing BSA (0.1 mL) [D(n)] are added to the bottom of a spectrophotometer cuvette and the enzyme mixture (20 mL) is added and mixed and the solution incubated at 30 C for 30 min.
Recovery.-All values reported in this study are for the monosaccharide in the hydrated form. In starch, for example, glucose is in the anhydro form (MW ¼ 162), thus hydrolysis of 100 g of starch yields $110 g of glucose. Presenting the carbohydrate as the weight of the particular monosaccharide simplifies subsequent calculations of the glycemic load of the particular food.
For accurate measurement of available carbohydrates, the sucrase/b-galactosidase mixture must give complete hydrolysis of sucrose (with no hydrolysis of fructan or FOS; Figure 1) and of lactose. The quantitative hydrolysis and recovery of sucrose and lactose by the enzymes used in the available carbohydrates method is shown in Figure 4. Sucrose or lactose (500 mg) were dissolved in maleate incubation buffer (20.5 mL) and then aliquots (0.2-1.0 mL) were transferred to 25 mL of water and the volumes of each adjusted to 26 mL. Aliquots of this solution were incubated with sucrase/maltase/b-galactosidase as per Figure 2020.07D and the amounts of glucose and fructose or glucose and galactose were measured spectrophotometrically, and  the amounts of sucrose or lactose were determined (allowing for the molecular weight of these disaccharides). Results are shown in Figure 4 as sucrose and lactose. The recovery of sucrose or lactose across the five concentrations employed (equivalent to 100-500 mg/incubation) was 100%, demonstrating complete hydrolysis of these two disaccharides at the concentrations of the sucrase and b-galactosidase used in the assay. In order to determine the effect of matrix on recovery of sucrose, lactose, or digestible starch, a variety of sample spiking experiments were performed. Six typical food samples were spiked with two levels of sucrose and lactose (5% w/w and 15% w/w) and of regular maize starch (10% w/w and 30% w/w), subjected to the complete incubation procedure and analyzed for galactose, glucose, and fructose. Concurrently, samples containing no spike were incubated and values obtained for galactose, glucose, and fructose obtained for these samples were subtracted from those obtained with the spiked sample. The data in Table 2 shows the recoveries obtained for galactose, glucose, and fructose were within 95-105% (w/w) for each of the samples studied, except for butter beans, in which case the recovery of the glucose component of both sucrose and lactose was $90% (w/w). The spiking experiments with butter beans and sucrose at 5% (w/w) were repeated numerous times and the recovery of the fructose was 98-104%, but the value for glucose ranged between 71 and 124%. It is considered that the high variation in recovery of the glucose component of the sucrose or lactose, but not the fructose or galactose of these added sugars, in butter beans samples is due to the fact that the glucose from the added sucrose or lactose is small compared to the glucose produced on hydrolysis of the starch in the sample. High absorbance values are obtained for the sample and the sample plus spike and the difference between the two absorbance values is small, leading to larger variations in the determined glucose value for the sucrose or lactose spike.
LOD and LOQ.-LOD (the lowest level at which detection of the analyte becomes problematic) and LOQ (the lowest level at which the performance of the assay is acceptably repeatable) of the available carbohydrates assay method were calculated by performing replicate assays (n ¼ 16) on a sucrose/cellulose sample with a relatively low content of available carbohydrates (5.17%, w/w). The analysis was performed as described except that the sample removed was added to 5 mL of water (instead of 25 mL) to obtain significant absorbance values in the assay. The LOD was 0.054% (w/w) using LOD ¼ 3 Â s 0 0 , where s 0 0 ¼ the SD of replicate measurements. The LOQ was calculated as 0.179% (w/w) using LOQ ¼ k Q Â s 0 0 , where s 0 0 ¼ the SD of replicate measurements. According to The International Union of Pure and Applied Chemistry, the default value for k Q ¼ 10.
Trueness (Bias).-Accuracy of the available carbohydrates assay method was assessed by comparison of the mean available carbohydrates content obtained for suitable reference materials with a specific reference value. There are no official CRMs for available carbohydrates, so the reference materials used for this analysis were a sucrose/cellulose mixture and a lactose/cellulose mixture.
Relative bias is calculated as follows: where b(%) ¼ relative bias; x ¼ mean available carbohydrates content; and x ref ¼ specific reference value. The accuracy of the available carbohydrates assay method is extremely high with a calculated relative bias of 1.7% for the lactose/cellulose mixture and 3.4% for the sucrose/cellulose mixture.
Precision.-The precision of the available carbohydrates assay method was assessed using eight food samples. For each sample, duplicate extractions were processed and analyzed by the available carbohydrates assay on four separate occasions by a single analyst. The available carbohydrates content of the samples tested covered a working range of 44.1-88.9% (w/w) available carbohydrates. The repeatability (RSD r , %) across this sample set was extremely high ( Table 3). The inter-day repeatability (RSD r , %) was less than or equal to 3.58% (w/w). This level of repeatability indicated a very high level of precision showing that the method is robust, reliable, and repeatable, and thus suitable for the application of measuring available carbohydrates in food and vegetable samples.
The available carbohydrates method has been applied to the measurement of carbohydrates in a wide range of cereals and cereal based foods, dairy products, food, and vegetables and the results are shown in Table 4. Determinations were performed in duplicate.
Stability.-The Available Carbohydrates Assay Kit as formulated by Megazyme comes with a two-year stability guarantee from date of purchase. When dissolved, the PAA pancreatic aamylase/amyloglucosidase [D(d)] must be stored on ice and used on the day of preparation. However, if prepared as an ammonium sulphate suspension, it is stable at 4 C for 3 months. All other kit components, if stored as recommended in the kit booklet, are stable for more than 2 years. Individual kit components may have longer stability guarantees: this information is available on the component label as the expiry date. Regular quality control testing is performed within the Megazyme QC laboratory.

Discussion
Available carbohydrates have been defined (1,2) as the sum of sugars (glucose, fructose, galactose, sucrose, maltose, and lactose) and complex carbohydrates ("malto" dextrins, starch, and glycogen). Historically, these have been measured individually by a combination of enzymatic, complex chemical (2), and HPLC procedures and the values pooled. The method described here involves the complete and specific hydrolysis of each carbohydrate to the component monosaccharides, glucose, fructose, and galactose and specific enzymatic measurement of these in a single reaction cuvette. With the increased knowledge of starch hydrolysis in the human small intestine and the recognition of the importance of resistant starch as a component of dietary fiber, it has become important to specifically measure digestible starch rather than total starch for the calculation of available carbohydrates. Consequently, food digestion in the small intestine has been simulated using a combination of gentle shaking or stirring in the presence of PAA/AMG at 37 C for 4 h, as is employed in AOAC Method 2017.16 (7,8). A sample of the incubation solution is removed, diluted in water, and an aliquot centrifuged. This solution is used in the determination of available carbohydrates. In the incubation with PAA and AMG, maltose, maltodextrins, glycogen, and digestible starch are hydrolyzed to D-glucose (with trace levels of maltose). On subsequent incubation with b-galactosidase (13), sucrase, and maltase, lactose is hydrolyzed to glucose and galactose, sucrose is specifically hydrolyzed to glucose and fructose and remaining traces of maltose are hydrolyzed to glucose ( Figure  2020.07D). Incubation with oligo-1,6-a-glucosidase gives complete hydrolysis of isomaltose (derived from IMO) to glucose. Galactose, glucose, and fructose are then measured enzymatically, as shown in Figure 2020.07E. Traditionally, sucrose has been hydrolyzed using invertase (b-fructofuranosidase), but this enzyme also acts on FOS resulting in   overestimation of the sucrose (Figure 1). Clearly, invertase is not suitable for the specific hydrolysis of sucrose in the presence of FOS The b-galactosidase employed here is MZ104 b-galactosidase (Megazyme) which has optimal activity at pH 6.5 which makes it ideal for use in conjunction with sucrase which has a similar pH optimum. The available carbohydrates content of a number of food materials is shown in Table 4. The repeatability of the available carbohydrates method has been determined by analyzing eight food materials in duplicate over 4 days (Table 3). Inter-day repeatability is excellent, with RSD r values ranging from 1.6 to 3.58.
The currently described "available carbohydrates" method together with the total dietary fiber method (AOAC Method 2017.16) (8) allows the measurement of all carbohydrates, including digestible and resistant starch, in food materials. For food labeling in the United States, the only approved method for labeling of carbohydrates in the nutrition facts panel involves subtracting the amount of crude protein, total fat, moisture, and ash from the total sample weight. Dietary fiber is included in the total carbohydrate value, but it can also be reported separately on the label. "Net carbohydrates" is determined by subtracting the dietary fiber value from the total carbohydrates value. A major problem in using this Galactose, g/100 g b "as is" Glucose, g/100 g b "as is" Fructose, g/100 g b "as is" Available carbohydrates, g/100 g b "as is" Available carbohydrates, g/100 g b "dwb" c Mean þ 2 SD approach is that of accumulated errors (4). In this paper, a simple method is described for the direct measurement of the available carbohydrates (glucose, fructose, galactose, sucrose, lactose, isomaltose, maltodextrins, and digestible starch) by direct measurement of glucose, fructose, and galactose. Such values also allow estimation of the glycemic index value of a food.

Conclusion
The method outlined in this document is a robust, quick, and easy method for the direct measurement of digestible carbohydrates in a broad range of human and animal foods and feeds. Data presented in this report verifies and validates that this method is fit for the purpose intended.